(1) Field of the Invention
The present invention relates to novel derived microorganisms and to a method for producing the derived microorganisms which contain foreign DNA encoding for nisin production. In particular, the present invention relates to derived bacteria containing foreign DNA encoding for nisin production.
(2) Prior Art
Nisin
The Merck Index (8th Edition) at page 6375 generally characterizes nisin as a polypeptide antibiotic produced by Streptococcus lactis, citing various publications including Mattick, Hirsch, Nature 154, 551 (1944); Lancet 250, 417 (1946); and 253, 5 (1947); Berridge et al., Biochem. J. 52, 529 (1952); and U.S. Pat. No. 2,935,503 (1960). The chemical structure is indicated to contain 34 amino acid residues, eight of which are rarely found in nature, including lanthionine (two alanines bonded to sulfur at the beta-carbons) and beta-methyllanthionine as described by Gross, J. Am. Chem. Soc. 93, 4634 (1971). Nisin is indicated to form crystals from ethanol and to be soluble in dilute acids. It is stable to boiling in acid solution. The Merck Index indicates that nisin is used in food processing and as a preservative, especially for cheese and canned fruits and vegetables.
A chemical structure of one form of nisin has been described by Gross and Morrell of the National Institute of Health in Chem. and Eng. News Page 18 (Sept. 24, 1973). The structure includes alpha-beta unsaturation in amino acids near the terminal amino and acid groups. It is speculated that the activity of nisin is related to reaction of the unsaturated amino acids with the sulfhydryl groups of enzymes in the affected microorganisms. Nisin generally has a published molecular weight range between about 6800 and 7500. Nisin is classed as an antibiotic produced by N-group lactic acid producing Streptococci. The inventors prefer the phrase "inhibitory substance" to "antibiotic" where nisin is generated in situ in a food by the microorganism, particularly by bacteria.
Use in Foods
Nisin in foods inhibits clostridial spoilage which is a major problem resulting from food storage. In addition, nisin inhibits psychotrophic bacteria which are particularly a problem with refrigerated foods. Thus nisin inhibits Streptococci of Groups A, B, E, F, H, K, M and N, Staphylococci, Micrococcus, Bacillus (some species) Clostridium, Mycobacterium, Lactobacillus, Octinomyces and Erysipelothrix. Nisin is particularly effective where the food has been partially heat treated. It is not affected by the presence of foods containing blood serum or milk. It is thus useful in settings where these substances are present in substantial amounts. At this time nisin can not be added to foods in the United States but is used in many countries elsewhere in the world; however, approval is being sought in the United States. It should be noted that nisin occurs naturally in fermented food products where nisin producing strains of Streptococcus lactis are present, particularly in milk products. Use in foods is described by Reddy et al J. of Food Science 35, 787-791 (1970) and in Reddy et al J. Food Science 40, 314-318 (1975). An assay procedure in foods is described by Trainer, J., et al J. Sci Fd. Agriculture 15 522-528 (1964). The Assay of Nisin In Foods. Fowler, G. G.; Garvis, B.; Tramer, J., Aplin & Barret Ltd., Yeovil, Somerset, U.K. Technical Series. Society for Applied Bacteriology, 1975, No. 8 pp 91-105.
Strains
Various strains of Streptococcus lactis are known to produce nisin, but S. cremoris and S. lactis subspecies diacetilactis do not. Such strains are described in: McClintok, H. et al. J. Dairy Research 19, 187-193 (1952); Campbell, L. L. et al Food Tech 13: 462-464 (1959); Hurst J. Gen Microbiology 44: 209-220 (1966); Mattick and Hirsch 12th Intern. Dairy Congress 2 (Sect 3) 546-550 (1949); Campbell et al, Food Preservation by Use of Chemicals 110-119 (about 1960); McClintock et al, J. Dairy Res 19: 187-193 (1952) (French); Rayman, Applied and Environmental Tech. 41: 375, 380 (1981); Scott et al Journal of Food Science 40: 115-126 (1981); and in U.S. Pat. Nos. 2,935,503; 3,093,551; 2,785,108; and 3,295,989.
Various technical articles have appeared describing nisin producing Streptococcus lactis strains. Included are Geis A, et al Applied Environmental Microbiology 45: 205-211 (1983); Kozak et al J. of Gen. Microbiology 83: 295-302 (1974); Pack, et al, J. Bacteriology 149 420-425 (1982); McKay, L. et al Applied and Environmental Microbiology 40: 84-91 (1980); Hurst A, J. Gen Microbiology 44: 209-220 (1966); Scherwitz, K., et al., Applied and Environmental Microbiology 45: 1506-1512 (1983); LeBlanc, D., et al., J. of Bacteriology 137: 878-884 (1979) and LeBlanc D., et al in Plasmids and Transposons. Environmental Effects and Maintenance Mechanisms, Edited by Colin Stuttard and Kenneth R. Rozee, Academic Press, 31-41 (1980). Some of these publications describe the plasmids in Streptococcus lactis and in particular a 28 Mdal plasmid which may encode for nisin production; however, none of the publications describe the transfer of the nisin encoding plasmid to non-nisin producing recipient bacteria or to other microorganisms.
A summary regarding nisin appears in Hurst, A., Advances Applied Microbiology 27: 85-123 (1981). This publication describes what is generally known about nisin.
A major problem described in the prior art is the phage susceptibility of nisin producing strains of Streptococcus lactis. Another problem is that the nisin is expressed at relatively low levels in the naturally occurring strains of Streptococcus lactis and the strains are difficult to grow. Still another problem is that nisin alone has some limitations of inhibitory activity against food spoilage microorganisms.
Objects
It is therefore an object of the present invention to provide nisin producing derived microorganisms containing foreign DNA which encodes for nisin production. It is further an object of the present invention to provide a large collection of nisin producing derived microorganisms whic have unrelated phage susceptibilities and which generate relatively large amounts of nisin. Also, provided are nisin producing derived microorganisms which can be adapted to become phage insensitive derivatives by direct challenge with phage for selection of insensitive clones without loss of desired functional characteristics, particularly nisin production. Further still it is an object of the present invention to provide nisin producing derived microorganisms which can be combined with other inhibitory substance producing microorganisms. These and other objects will become increasingly apparent by reference to the following description.